Although, acute inflammatory responses have a protective role in innate immunity and host defense, dysregulation and failure to properly resolve these responses cause inflammatory and infectious disease. Several key cytosolic pattern recognition receptors signal through proteins containing the Caspase recruitment domain (CARD), which is a protein-protein interaction domain, leading to inflammasome and NF-kB activation, and inflammatory cytokine and type I interferon (IFN) production. However, the molecular mechanisms by which these pathways are controlled to limit excessive and detrimental inflammatory responses, are largely unknown. Three small proteins containing only a CARD, which are referred to as CARD-only proteins (COPs) exist in humans, but like the related PYRIN domain-only proteins (POPs), are lacking from mice, which supports the notion that these inflammatory responses require a tight control in particular in humans. COPs have been identified over a decade ago, but except of a few initial overexpression studies, nothing is known about the precise role of these proteins. We demonstrated key inflammasome regulatory activities of the three POPs encoded in humans using a comprehensive analysis in humans and transgenic mice, and we hypothesize that similarly, COPs will have an important role in regulating inflammatory responses and our preliminary data indicate that COPs have a broader role than POPs by targeting several key pattern recognition receptors. The research outlined in this proposal is geared to define the precise role of each COP in macrophages during infection and inflammation, using a comprehensive analysis combining biochemical, molecular and genetic approaches. We will also define their role during inflammatory and infectious disease, using novel generated transgenic mice for all three COPs. We expect that our research will uncover novel molecular control mechanisms that prevent inappropriate inflammation and will therefore be highly significant and relevant for better understanding innate immunity and inflammatory disease and for providing the basis for developing novel therapies to benefit patients and will therefore positively affect human health.